The limited proliferative ability of normal animal cells in culture is widely accepted as a manifestation of aging at the cellular level. Most of the investigations of this phenomenon have been devoted to the study of intracellular changes that occur during in vitro aging. However, it is likely that an understanding of the mechanisms leading to the inevitable and essentially irreversible loss of proliferative ability in normal cells will have significance for a large number of biological problems, including the processes of neoplasia and abnormal development. Recent experimental evidence from cell hybridization and cell reconstruction experiments has led to the idea that after a period of active growth in culture normal cells produce a specific substance(s) that prevents DNA synthesis and cell division, thereby resulting in what has been commonly called cellular senescence. We have further found that human diploid fibroblasts held in a nondividing state (quiescent cells) produce an inhibitory substance(s) similar to that produced by senescent cells. Proteins that block the initiation of DNA synthesis can be extracted from the membranes of these cells and, when poly(A)+ mRNA from senescent cells is microinjected into young cells, the initiation of DNA synthesis is blocked. We plan to isolate and identify cDNA sequences homologous to the genomic DNA coding for the inhibitor of DNA synthesis found in senescent human diploid fibroblast (HDF) cells. We can then identify the genomic DNA sequences, examine the structure of the gene and sequences flanking the gene to determine the regulatory sequences, and determine if changes occur in the gene and the flanking regions during in vitro aging. We will then begin to study the mechanism of action of the senescence cell inhibitor. We will determine whether the inhibitor sequences found in senescent cells are the same as those expressed in quiescent cells. If they are, we will study the modulation of gene expression in quiescent cells by addition and removal of growth factors. We will also investigate the ability of activated oncogenes and proto-oncogenes to overcome the action of the inhibitor. We will probe the premature aging syndrome and DNA repair-deficient cells of R. Moses with DNA probes and with antibodies to the senescence facto to determine whether the timing and amount of expression differs from that occuring in cells derived from normal individuals.